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Nicotinamide effects on adiposity, energy metabolism, inflammation and atherosclerosis in mice
Karen Alejandra Méndez Lara
Autonomous University of Barcelona
Department of Biochemistry and Molecular Biology.
Doctoral Program in Biochemistry, Molecular Biology and Biomedicine.
Nicotinamide effects on adiposity, energy metabolism, inflammation and atherosclerosis in mice
Thesis presented by Karen Alejandra Méndez Lara to apply for the degree of Doctor by the Autonomous University of Barcelona in the program of PhD. in Biochemistry, Molecular Biology and Biomedicine.
This thesis has been carried out in the Molecular Bases of Cardiovascular Disease group of the Hospital de Santa Creu i Sant Pau, co-directed by Dr.
Josep Julve and Dr. Francisco Blanco-Vaca.
Dr. Josep Julve Senior Researcher
Institut de Recerca de l’Hospital de la Santa Creu i Sant Pau
Dr. Francisco Blanco Vaca
P.I. of Molecular Bases of Cardiovascular Disease group.
Servei de Bioquímica, Hospital de la Santa Creu i Sant Pau.
Departament de Bioquímica i Biologia Molecular.
Universitat Autònoma de Barcelona (UAB).
Karen Alejandra Méndez Lara
Institut de Recerca de l’Hospital de la Santa Creu i Sant Pau
Barcelona, September 2019
TABLE OF CONTENTS
TABLE OF CONTENTS ...5
TABLE OF FIGURES ... 11
TABLE OF TABLES ... 17
ABBREVIATIONS ... 19
ABSTRACT ... 21
GRAPHICAL ABSTRACT ... 25
1. INTRODUCTION ... 27
▶
1.1 Mouse models of obesity... 30
▶
1.2 Adipose tissue plasticity in physio(patho)logy ... 32
1.2.1 General characteristics of adipose tissue ... 33
1.2.2 Types of adipose tissue ... 33
1.2.3 Functional units of fat pads ... 38
1.2.4 Dysfunctional adipose tissue in obesity ... 42
▶
1.3 Obesity and cardiovascular disease ... 50
▶
1.4 Anti-obesity strategies ... 51
1.4.1 Current emerging therapeutic strategies targeting adipose tissue ... 51
2. HYPOTHESIS ... 61
OBJECTIVES ... 63
3. MATERIALS AND METHODS ... 65
▶
3.1 Mice and diets ... 65
3.1.1 Common methods and procedures in different experimental designs... 65
3.1.2 Evaluation of the safe dose ... 66
3.1.3 Evaluation of vitamin B3 forms on body weight gain prevention 69 3.1.4 Assessment of anti-atherogenic action by NAM ... 72
3.1.5 Diets used in this work ... 73
▶
3.2 Biochemical analyses ... 74
3.2.1 Plasma analysis of biochemical parameters ... 74
3.2.2 Cytokine determination in plasma using a multiplex approach ... 83
3.2.3 Isolation of lipoproteins from sequential ultracentrifugation ... 84
3.2.4 ApoE-deficient mice phenotyping ... 93
3.2.5 Nicotinamide and N-methylnicotinamide determination in plasma ... 93
▶
3.3 Tissue analyses ... 96
3.3.1 Tissue collection ... 96
3.3.2 Histological analysis ... 96
3.3.2a Adipocytes, BAT and liver droplets quantification ... 96
3.3.2b Analysis and quantification of arterial lesions ... 97
3.3.2c ORO Staining ... 97
3.3.2d F4/80 Immunohistochemical staining ... 99
3.3.3 Determination of fecal and liver triglycerides... 100
3.3.4 Determination of energy metabolites ... 100
3.3.5 Protein abundance and gene expression analyses ... 103
3.3.5a Western blot analysis ... 103
3.3.5b Gene expression analysis ... 104
▶
3.4 Functional tests ... 110
3.4.1 Glucose tolerance test ... 110
3.4.2 Insulin sensitivity test ... 111
3.4.3 Susceptibility to lipoprotein oxidation ... 112
▶
3.5 In vivo kinetic studies ... 114
3.5.1 In vivo non-HDL turnover ... 114
3.5.2 Distribution of intragastrically-administered [3H]-cholesterol in mice ... 118
3.5.3 Assessment of oxygen consumption in mouse by indirect calorimetry ... 119
3.5.4 Magnetic resonance imaging (MRI) analysis ... 120
3.5.5 Palmitate -oxidation measurement in isolated mitochondria ... 121
▶
3.6 In vitro studies ... 124
3.6.1 NAM-mediated prevention of lipopolysaccharide(LPS)-induced inflammation ... 124
▶
3.7 Statistical analysis ... 125
4. RESULTS ... 127
4.1 Evaluating vitamin B
3forms tolerance ... 127
4.1.1 Choosing the best dose ... 127
4.2 Impact of vitamin B
3forms on body weight and adiposity in
non-obese mice fed a regular diet (RD) ... 132
4.2.1 Gross parameters and systemic phenotype ... 132
4.2.1a Biochemical parameters ... 139
4.2.1b Glucose homeostasis ... 141
4.3 Impact of vitamin B3 forms on body weight and adiposity in a mouse model of diet-induced obesity (DIO) ... 144
4.3.1 Gross parameters and systemic phenotype ... 144
4.3.1a Biochemical and molecular parameters ... 150
4.3.1b Glucose homeostasis ... 153
4.4 Impact of different doses of NAM on body weight and adiposity in DIO mice ... 155
4.4.1 Gross parameters and systemic phenotype ... 155
4.4.1a MRI analysis ... 158
4.4.1b Biochemical parameters ... 161
4.4.1c Histologic and molecular analysis on fatty liver ... 165
4.4.1d Glucose homeostasis ... 169
4.4.2 Energy metabolism ... 171
4.4.2a Global energy expenditure by indirect calorimetry analysis .... 171
4.4.2b Energy metabolites in scWAT ... 176
4.4.2c Mitochondrial activity and mass surrogates in iBAT ... 180
4.4.3 Molecular and plasticity analysis on adipose tissue ... 182
4.4.3a Browning ... 182
4.4.3b Obesity-related inflammation ... 190
4.5 Effect of NAM-treated ApoE-deficient mice on chronic
inflammation: Atherosclerosis ... 193
4.5.1 Inflammation analysis in vitro ... 193
4.5.2 Gross parameters and systemic phenotype ... 194
4.5.2a Biochemical parameters ... 196
4.5.2b Non-HDL metabolism ... 198
4.5.2c Anti-oxidative function of lipoproteins ... 201
4.5.2d Histologic and molecular analysis on fatty liver ... 206
4.5.3 Atherosclerosis development ... 207
4.5.4 Systemic inflammation markers ... 209
5. DISCUSSION ... 215
5.1 Determination of oral dose of NAM to mice ... 215
5.2 Anti-obesity mechanisms of NAM ... 219
5.3 Effect of NAM on adipose “browning” ... 220
5.4 Contribution of NAM-mediated NAD+ boost to adipose phenotype ... 224
5.5 Effect of NAM on other mechanisms of energy expenditure 226 5.6 NAM on homeostasis and metabolism of glucose .... 227
5.7 NAM on fatty liver ... 228
5.8 NAM administration prevented vascular chronic inflammation and atherosclerosis in ApoE-deficient mice ... 229
5.8a Anti-inflammatory effect of NAM ... 229
5.8b Anti-oxidant effect of NAM... 232
5.9 Limitations of the study ... 235
6. CONCLUSIONS ... 237
7. References ... 239
TABLE OF FIGURES
Figure 1 Factors that affect obesity development ... 28
Figure 2 Fat depot distribution in humans and mice. ... 35
Figure 3 Morphological appearance of BAT and WAT. ... 36
Figure 4 Distribution of major BAT depots in mouse and human. ... 37
Figure 5 Modulation of immune-metabolism during obesity. ... 41
Figure 6 Central nodes for cellular NAD+ metabolism ... 54
Figure 7 Structural formula of nicotinamide ... 58
Figure 8 Choosing a safe dose of vitamin B3 forms. ... 67
Figure 9 Mouse testing using higher doses of NAM than 1%. ... 68
Figure 10 Validation of the maximal safe dose. ... 68
Figure 11. Impact of vitamin B3 forms on mice phenotype. ... 69
Figure 12. Impact of vitamin B3 forms on a mouse model of induced obesity. ... 70
Figure 13. Impact of different doses of NAM on a mouse model of induced obesity. ... 71
Figure 14. Effect of NAM in the development of atherosclerosis. ... 72
Figure 15. Serial dilutions of the standard. ... 77
Figure 16. Serial dilutions of the standard. ... 80
Figure 17. Radding and Steinberg formula. ... 85
Figure 18. Effect of NAM on the production of TNFa by LPS-stimulated macrophages. ... 125
Figure 19. Effect of vitamin B3 forms on body and liver weight. ... 127
Figure 20. Effect of vitamin B3 forms on body weight gain. ... 131 Figure 21. Effect of vitamin B3 forms on body weight gain. ... 131 Figure 22. Body weight follow-up in mice fed a RD. ... 132 Figure 23. Relation between body weight and adiposity with adipokines related with obesity. ... 136 Figure 24. Effect of vitamin B3 forms on gross parameters in mice fed a RD.
... 137 Figure 25. Relationship between total body and fat pad weights. ... 137 Figure 26. Effect of vitamin B3 forms on fat cell size from eWAT and scWAT in mice fed a RD. ... 138 Figure 27. Relationship between total body and fat pad weights and plasma levels of FFA. ... 140 Figure 28. Functional analysis of glucose metabolism in mice on a RD: GTT.
... 142 Figure 29. Functional analysis of glucose metabolism in mice on a RD: insulin sensitivity. ... 143 Figure 30. Effect of vitamin B3 forms on DIO mice. ... 146 Figure 31. Relation between body weight and adiposity with adipokines involved in obesity in DIO mice. ... 148 Figure 32. Histological images of epididymal and subcutaneous WAT in DIO mice. ... 149 Figure 33. Gene expression of molecular targets involved in inflammatory processes in eWAT in DIO mice. ... 149 Figure 34. Relationship between total body and fat pad weights and plasma levels of FFA in DIO mice. ... 151 Figure 35. Gene expression of Cd36 in liver in DIO mice. ... 152 Figure 36. Functional analysis of glucose metabolism in DIO mice: GTT. .... 154
Figure 37. Functional analysis of glucose metabolism in DIO mice: insulin sensitivity. ... 154 Figure 38. Effect of different doses on NAM on gross parameters in DIO mice.
... 157 Figure 39. Relationship between total body and fat pad weights in DIO mice.
... 158 Figure 40. Representative T1W used to measure total fat pad in DIO mice and treated with different doses of NAM for 3 consecutive months. ... 159 Figure 41. . Box-plot representing relative values or ROIs showing total fat pad and liver fat in DIO mice and treated with different doses of NAM for 3 consecutive months. ... 160 Figure 42. Relationship between total body and fat pad weights with liver fat fraction in DIO mice. ... 161 Figure 43. NAM and its methylated form (me- NAM) displayed a dose-dependent shape in plasma in DIO mice. ... 162 Figure 44. Relationship between total body and fat pad weights and plasma levels of FFA in DIO mice. ... 163 Figure 45. Effect of NAM on fatty liver in DIO mice. ... 165 Figure 46. Impact of NAM over fatty liver-related markers in DIO mice. ... 167 Figure 47. Gene expression of molecular targets involved in NAM metabolism in liver and adipose tissue in DIO mice. ... 169 Figure 48. Effect of different doses of NAM on functional analysis of glucose metabolism in DIO mice: glucose tolerance test. ... 170 Figure 49. Effect of different doses of NAM on functional analysis of glucose metabolism in DIO mice: insulin sensitivity ... 170 Figure 50. Analysis of energy expenditure of DIO mice measured in metabolic cages. ... 172
Figure 51. Analysis of glucose oxidation measured in metabolic cages of DIO
mice. ... 173
Figure 52. Analysis of lipid oxidation measured in metabolic cages of DIO mice. ... 174
Figure 53. Relationship between total body and fat pad weights with RER in DIO mice. ... 175
Figure 54. Analysis of NADt, NAD+, NADH and NAD+/NADH in scWAT in DIO mice. ... 176
Figure 55. Relationship between NAD+ and NADt and Nampt mRNA in scWAT in DIO mice. ... 177
Figure 56. Analysis of [ATP+ ADP+ AMP] and AMP/ATP ratio in scWAT in DIO mice. ... 178
Figure 57. Relationship between [ATP+ADP+AMP] and [NAD+/ NADH] ratio in scWAT in DIO mice. ... 178
Figure 58. AMPK analysis in scWAT of DIO mice. ... 179
Figure 59. AMPK activity and its relationship with adenosine phosphate in subcutaneous white adipose tissue. ... 180
Figure 60. Analysis of mitochondrial activity in BAT of DIO mice. ... 180
Figure 61. Western blot analysis of Complex III and TIM44 protein in iBAT of DIO mice. ... 181
Figure 62. Effect NAM over browning on scWAT in DIO mice. ... 183
Figure 63. Effect NAM on iBAT in DIO mice. ... 183
Figure 64. Analysis of Ucp1 protein in scWAT of DIO mice. ... 184
Figure 65. Western blot analysis of Ucp1 protein in BAT of DIO mice. ... 185
Figure 66. Relationship mRNA levels of browning and mitochondrial targets with RER in scWAT of DIO mice. ... 189
Figure 67. Effect NAM over crown-like structures (CLS) on eWAT in DIO mice. ... 190
Figure 68. Evaluation of plasma cytokines associated with inflammation in DIO mice. ... 192 Figure 69. Gene expression of an inflammation target in an in vitro assay. .. 193 Figure 70. Effect of vitamin B3 forms on gross parameters in ApoE-deficient mice. ... 194 Figure 71. NAM and its methylated form (me-NAM) displayed a dose-dependent shape in plasma in ApoE-deficient mice. ... 196 Figure 72. Effect of NAM on metabolic fate of non-HDL lipoproteins in plasma in ApoE-deficient mice. ... 200 Figure 73. Atheroprotective property of non-HDL particles of ApoE-deficient mice. ... 202 Figure 74. Antioxidant activity of LDL particles of human plasma incubated with different concentrations of NAM ... 203 Figure 75. Effect of NAM in liver in ApoE-deficient mice. ... 207 Figure 76. Representative images of proximal aorta of ApoE-deficient mice treated with different doses of NAM for 1 month. ... 208 Figure 77. Relationship between circulating plasma NAM levels and atherosclerosis in ApoE-deficient mice. ... 209 Figure 78. Evaluation of plasmatic cytokines associated with inflammation in ApoE-deficient mice. ... 210 Figure 79. Gene expression in thoracoabdominal aorta determined by real-time PCR analysis in ApoE-deficient mice. ... 211 Figure 80. Representative IHQ of F4/80 or thoracoabdominal aortas of ApoE- deficient mice treated and non-treated with NAM. ... 212 Figure 81. Gene expression of molecular targets regulated by LXR in the thoracoabdominal aorta of ApoE-deficient mice. ... 213
TABLE OF TABLES
Table 1. Experimental diet nutrient information. ... 73 Table 2. Transitions used in QqQ shown by different metabolites. ... 95 Table 3. List of specific Taqman probes used for gene expression analysis. 108 Table 4. List of primers sequences used for gene expression analysis. ... 109 Table 5. . Effect of vitamin B3 forms on hepatic transaminases in mice fed a RD.
... 128 Table 6. Effect of vitamin B3 forms on plasma biochemical parameters in mice fed a RD. ... 129 Table 7. Effect of vitamin B3 forms on plasma biochemical parameters in mice fed a RD. ... 130 Table 8. Effect of vitamin B3 forms on gross parameters in mice fed a RD .. 133 Table 9. Effect of vitamin B3 forms on critical hormones involved in adiposity in mice fed a RD. ... 135 Table 10. Effect of vitamin B3 forms on plasma biochemical parameters in mice fed a RD. ... 139 Table 11. . Effect of vitamin B3 forms on hepatic and renal parameters in mice fed a RD. ... 141 Table 12. Effect of vitamin B3 forms on glucose homeostasis in mice fed a RD.
... 141 Table 13. Effect of vitamin B3 forms on gross parameters in DIO mice. ... 145 Table 14. Effect of vitamin B3 forms on critical hormones involved in adiposity in DIO mice. ... 147 Table 15. Effect of vitamin B3 forms on biochemical parameters in DIO mice.
... 150 Table 16. . Effect of vitamin B3 forms on hepatic and renal parameters in DIO
Table 17. Effect of vitamin B3 forms on glucose homeostasis in DIO mice. . 153 Table 18. Effects of different doses of NAM in DIO mice. ... 156 Table 19. Effect NAM on plasma biochemical parameters in DIO mice. ... 163 Table 20. Effect of NAM on hepatic and renal parameters in DIO mice. ... 164 Table 21. Gene expression of molecular targets involved in macrophages infiltration and inflammation in DIO mice ... 166 Table 22. Gene expression of molecular targets involved in NAM metabolism in liver and adipose tissue in DIO mice. ... 168 Table 23. Gene expression of molecular targets involved in energy homeostasis and browning of scWAT and iBAT in DIO mice. ... 187 Table 24. Gene expression of molecular targets involved in macrophages infiltration and inflammation in DIO mice. ... 191 Table 25. Effect of NAM on gross parameters in ApoE-deficient mice. ... 195 Table 26. Effect of NAM on plasma biochemical parameters in ApoE-deficient mice. ... 197 Table 27.Effect of NAM on the relative composition of non-HDL particles of ApoE-deficient mice. ... 198 Table 28. Hepatic gene expression profile of molecular targets involved in non- HDL clearance in ApoE-deficient mice. ... 201 Table 29. Gene expression of molecular targets involved in oxidation and inflammatory properties in liver, eWAT and aorta of ApoE-deficient mice .... 204 Table 30. Gene expression of hepatic molecular targets involved in NAM metabolism of ApoE-deficient mice. ... 205 Table 31. Effect of NAM on hepatic and renal parameters in ApoE-deficient mice.
... 206
ABBREVIATIONS
ADP AKT
adenosin diphosphate protein kinase B
ALT alanine aminotransferase
AMP adenosin monophosphate
AMPK 5' adenosine monophosphate-activated protein kinase AST aspartate aminotransferase
ATP BAT
adenosin triphosphate brown adipose tissue cDNA
Cpt1b DIO
complementary deoxyribonucleic acid carnitine palmitoyltransferase 1b, muscle diet induced obesity
DNA deoxyribonucleic acid
EDTA Ethylenediamine tetraacetic acid ELISA enzyme-linked immunosorbent assay
eNampt extracellular nicotinamide phosphoribosyltransferase
FFA free fatty acid
FOXO GTT
the forkhead box O family glucose tolerance test HDL
HFD
high density lipoprotein high fat diet
HPLC IL-4 IL-6 IL-10
high-performance liquid cromatography interleukin 4
interleukin 6 interleukin 10 iNampt
ITT
intracellular nicotinamide phosphoribosyltransferase insulin tolerance test
LDL low density lipoprotein
LC-MS/MS liquid chromatography (LC) with mass spectrometry in tandem (MS/MS)
mRNA NA
Messenger ribonucleic acid nicotinic acid
NAD+ nicotinamide adenine dinucleotide, oxide NADH nicotinamide adenine dinucleotide, reduced
NAM nicotinamide
Nampt nicotinamide phosphoribosyltransferase NMN mononucleotide nicotinamide
NR riboside nicotinamide
P-AMPK phosphorylated 5' adenosine monophosphate-activated protein kinase
PCR PGC-
1a/Ppargc1a
polimerase chain reaction
Peroxisome Proliferator–Activated Receptor Gamma Coactivator alpha
Pparg PGC-
1b/Ppargc1b
peroxisome proliferator activated receptor gamma
peroxisome proliferative activated receptor, gamma, coactivator 1 beta
RNA ribonucleic acid
qPCR quantitative PCR
ROS reactive oxygen species
SD SEM SIRT1
standard deviation
standard error of the mean sirtuin 1
TCA Tnfa Ucp1 VLDL WAT
trichloroacetic Acid tumor necrosis factor'
uncoupling protein 1 (mitochondrial, proton carrier)' very low-density lipoproteins
white adipose tissue
ABSTRACT
Background and hypothesis
Adipose tissue dysfunction is a hallmark of obesity and is frequently associated with distorted metabolic homeostasis, cardiovascular and chronic, low-grade inflammatory diseases. Several recent studies point to pharmacological and/or nutritional health initiatives targeting adipose tissue being a promising approach to obesity prevention. In this regard, nicotinamide adenine dinucleotide (NAD)+
precursors, such as nicotinamide riboside and mononucleotide nicotinamide has been proven beneficial in increasing energy metabolism and preventing body weight gain in vivo. However, neither their favorable anti-obesity impact on disturbed white adipose tissue (WAT) physiology and plasticity nor in alleviating chronic inflammation, which frequently accompanies obesity, was not eventually pursued in any of these studies. In addition to the above-mentioned NAD precursors, nicotinamide (NAM) is also a physiological precursor of NAD+.
However, its contribution in boosting energy metabolism and body weight gain still remains elusive. Although a growing body of evidences also supports a role for NAM as an anti-oxidant and anti-inflammatory agent both in vitro and in vivo, its potential contribution in preventing atherosclerosis, which is one of the main mechanisms involved in cardiovascular disease in vivo, has not previously been proven yet.
Aims
The aim of this study was twofold: to investigate the effect of NAM supplementation in (1) preventing weight gain and adiposity; (2) improving features of chronic inflammation in appropriate mouse models of obesity (diet-induced obesity -DIO- mice) and atherosclerosis (i.e., ApoE-deficient mice).
Results
NAM administration to mice was provided orally via tap water at libitum. Its administration was shown palatable, safe and well tolerated at doses below 1%.
NAM supplementation, at the highest dose used (1%) (NAM HD-treated mice), prevented body weight gain, with the latter being mainly and repeatedly accompanied by reduction in fat accumulation in different regional depots, and hepatic steatosis. Mechanistically, such anti-adiposity effect by NAM was mainly accompanied by an [i] increased global energy expenditure, [ii] enhanced promotion of browning in subcutaneous (sc)WAT, as revealed by elevations in the relative mRNA and protein abundance of the uncoupling protein (Ucp)-1, and [iii] elevation of the de novo synthesis of NAD+ and NAD/NADH ratio in scWAT of NAM HD-treated, DIO mice. Notably, the AMP content was significantly elevated in scWAT of NAM HD-treated, DIO mice. Also, the NAD+/NADH ratio was directly related to the AMP/ATP ratio. Overall these data suggested a situation of energy demand in scWAT from NAM HD-treated mice.
Concomitantly, the protein abundance of the active (phosphorylated) form of AMP-activated kinase was also elevated in this tissue of NAM HD-treated mice.
NAM supplementation also improved the global inflammatory condition and prevented atherosclerosis development in mice. This was revealed by [i] elevations in the circulating concentrations of interleukin (IL-)10 and [ii] up- regulation of relative mRNA of Il10 in both adipose and aortic tissues, which potentially suggested a switch to anti-inflammatory M2 macrophages. This phenotype was accompanied by a commensurate reduction in atherosclerosis development in NAM-treated, ApoE-deficient mice. In addition to improved inflammation, non-HDL of NAM-treated, ApoE-deficient mice were less prone to oxidation than those from untreated mice, being this effect at least partly provided by the intrinsic anti-oxidant action of NAM.
Conclusions
Dietary supplementation with NAM to mice prevented body weight gain and adiposity by boosting energy expenditure, with this being mainly attributed to induction of browning and energy demand in scWAT. NAM also promoted anti- inflammatory and anti-oxidant actions. Its administration increased gene expression Il10 in target tissues, including aorta, and protected against development of atherosclerosis.
GRAPHICAL ABSTRACT
In brief:
This work shows that NAM supplementation via tap water prevents whole-body and fat gain in mice by boosting energy metabolism and inducing browning in subcutaneous (sc)WAT of obese mice. Also, in ApoE-deficient mice, NAM treatment promotes anti –inflammatory and anti-oxidant effects, being this associated with atherosclerosis prevention.
1. INTRODUCTION
Obesity is a main, yet most neglected, public health concern worldwide [1]. The epidemic of obesity presents a major challenge to chronic disease prevention and health across the lifespan in human beings. Steadily rising prevalence of overweight and obesity have increased in the last years manly due to an increasingly sedentary lifestyle and nutritional transition consumption to processed and hyper caloric foods [2]. This trend has driven a remarkable increase in our understanding of adipose biology and obesity.
Overweight or obesity is defined as the accumulation of excess fat in the body which usually appears when energy intake (i.e, food consumption) exceeds caloric expenditure (i.e., basal metabolism, which includes biochemical pathways required to maintain global metabolic homeostasis, physical activity and adaptive thermogenesis). Energy expenditure is along with glucose intolerance and insulin resistance a common metabolic alteration in obesity [3].
Obesity has not been considered until relatively recently as a complex chronic disease. In its development, this metabolic condition results from a constant and complex interplay between predisposing genes, socioeconomic, behavioral and environmental stimuli [4] (Figure 1). In this context, genome-wide association studies have discovered genes that affect appetite and adiposity [5]. Over- consumption of hyper caloric foods and sedentary lifestyle are the best characterized environmental factors involved in adipose mass gain. Ageing, gender, ethnicity are other factors that may also influence adiposity [5]. Some drugs, including thiazolidinediones (i.e., a class of PPAR agonists), anti- depressants and anti-convulsants, glucocorticoids, estrogens, atypical anti- psychotics, can contribute to fat accumulation.
Figure 1 Factors that affect obesity development
Adipose tissue plays an important role in the maintenance of metabolic homeostasis in vivo [6]. The storage of energy as lipids and sequester them from the extracellular environment in intracellular organelles known as lipid droplets is a highly conserved mechanism in mammals. Adipose tissue depots (also called fat pads) are found in different regions but they are also present in close contact with other organs, where they modulate organ remodeling and function.
During obesity, adipose tissue plasticity and expandability underlie tissue dysfunction and are key determinants of obesity-associated metabolic dysregulation [7]. These features frequently appear when adipose tissue fails to appropriately expand to store energy overload, which leads to adverse metabolic problems in ectopic fat accumulation in many other tissues for a long time. This phenotype further results in other health complications and reduces life expectancy [8], thereby increasing rates of morbidity and mortality in obese patients. However, adipose gain may not necessarily be pathogenic [9].
Compelling evidence support the concept that the pathogenic link between fat gain and obesity-related metabolic and cardiovascular co-morbidities may be rather related to fat function and ectopic accumulation.
Obesity is also a largely preventable disease. Current attempts have been moderately effective for the treatment of obesity [10]. Despite having been proved beneficial for treating obesity, the effectiveness of existing pharmacological and dietary approaches fails to be maintained at long term.
Thus, a better understanding of the etiology of obesity is required for the development of more successful and personalized prevention and treatment strategies. The latter have been suggested at least partly to be attributed to the fact that such therapies have not been directly addressed to target tissues which become physiologically disrupted. One of the most important representatives is the white adipose tissue. Adipose tissue is a multifactorial organ which becomes severely dysfunctional and fails to appropriately face to excess energy incomes during obesity. Indeed, this tissue has gained substantial interest with regard to its potential to be therapeutically targeted [10].
The rising incidence of obesity and costs to treat its associated diseases rises the need for a better understanding of all aspects of adipocyte (fat cell) biology, to ascertain how increased adiposity leads to disease. To that effect, mouse models have been an essential tool in expanding our understanding of molecular roots of obesity is a pre-requisite to improve both prevention and management of this entity. Studies in rodents and humans have allowed the identification of genes predisposing to obesity and analyzing the effect of targeted treatments using gene manipulation, pharmacological and nutritional approaches.
Importantly, the accessibility of target tissues involved in obesity that are not easily attainable from humans also contribute to further dissect the effectiveness of each of these therapies and anticipate to undesirable metabolic side-effects [11].
▶ 1.1 Mouse models of obesity
Obesity involves decades of pathophysiological changes and adaptation.
Therefore, it is difficult to ascertain the exact mechanisms for this long-term process in humans. Several surrogate mouse models are the most common experimental approaches to circumvent some of these issues.
Admittedly, the criteria for choosing animal models of obesity are based on altered control of eating and/or body weight. The availability of genetically defined strains as well as widely-developed genetic manipulation techniques allow to test the contribution of genes in the development of obesity [12]. This includes murine gene loss- or gain-of-function strategies, monogenic and polygenic models, and different environmental exposure models [13].
The mouse model of diet-induced obesity (DIO) has become one of the most important approaches for understanding the interplay of high-calorie diets in westernized societies and the development of obesity. The DIO model closely mimics the increasingly availability of nutritionally poor, high-calorie foods commonly consumed over the past decades, and which are main contributors of the obesity trend in human societies. High-fat diets produce increased body weight and increased adiposity in various obesity-prone mouse strains [14].
Obesity-prone and -resistant definition is based on body fat composition data from “Naggert1” in the ‘Mouse Phenome Database’ (MDP) (https://phenome.jax.org/projects/Naggert1), whereby body fat composition was measured in up to 39 different mouse strains that were fed a HFD for 8 weeks.
Importantly, DIO mice exhibit obesity and metabolic phenotypes that are comparable to humans and can be measured with standardized diagnostic tests.
Based on earlier reports [15, 16], the obesity-prone, inbred strain C57BL/6J is commonly selected to study the obesity-promoting effects of a HFD. General advantages of working with mouse models include the low maintenance cost, which is related to its small size and the fact that they reach sexual maturity more rapidly than other mammals. Moreover, mice breeding is easy as they have a shorter gestation period and relatively abundant offspring to conduct studies.
More detailed phenotyping, such as direct metabolic measurements and
assessment of body fat content, that are difficult and costly in large numbers of humans are also possible in mice [17-19]. Importantly, environmental factors can be carefully controlled and specifically manipulated in mouse models [20], thereby reducing environmental heterogeneity. Mice also provide obesity-related tissues such as hepatic tissue that are otherwise difficult to obtain in humans [21]. However, mouse models of obesity also show some flaws. For instance, different conclusions may be reached in mouse and humans due to the analysis of different phenotypes. Indeed, body mass index (BMI) measurements are typical in human studies, whereas the direct measurement of percent of body fat or body fat mass is more common in mouse models. Unlike humans, there is no defined threshold for obesity based on BMI in mice. Also, and compared with humans, the secondary complications of obesity substantially depend on the genetic background of the strain [22].
▶ 1.2 Adipose tissue plasticity in physio(patho)logy
Adipose tissue is designed to function as the main long-term, energy-reservoir organ, and actively controls energy homeostasis. Indeed, this tissue stores excess energy in the form of triglycerides in living lipid-laden, parenchymal cells, defined as adipocytes, and is able to mobilize lipid reserves by releasing free fatty acids under conditions of nutritional deprivation or caloric restriction [5].
The classical perception of adipose tissue as an energy store has been replaced over the last decades by the notion that this tissue also plays a central role in lipid and glucose metabolism and also serves as an endocrine organ capable of synthesizing a number of biologically active compounds, including hormones and cytokines, that signal metabolic homeostasis of adipose tissue and also other organs [23, 24]. Particularly, leptin and adiponectin are secreted by adipose tissue and finely modulate feeding behavior and glucose and lipid metabolism, as well as many other physiological processes such as inflammation, angiogenesis or energy homeostasis [23, 25]. As a rule, plasma adipokines rise proportionally with fat mass, except for plasma adiponectin which is frequently decreased in obese patients [23].
Given its lipid buffering capacity and its endocrine function, healthy adipose tissue regulates circulation lipids flux according to energy demands controlling energy intake and expenditure. Thus, adipose tissue of healthy, non-obese individuals is able to maintain energy homeostasis, systemic insulin sensitivity and prevent overfeeding [26], but during obesity, it becomes dysfunctional and fails to confer such beneficial metabolic effects.
In mammals, adipose tissue is also relevant in thermoregulation through its insulator properties and ability to generate heat via non-shivering thermogenesis.
Adipose tissue and inflammation are also closely related in obesity [27]. Indeed, adipose tissue undergoes fibro-inflammation, which further compromises its functionality. During sustained obesity the enlargement (hypertrophy) of adipocytes is frequently accompanied by macrophage/leukocyte infiltration in
both humans and rodent models [28]. Inflammatory cell recruitment in adipose tissue is further linked to systemic inflammation and oxidative stress [29].
Importantly, pro-inflammatory cytokines, but also chemokines and adipokines are then secreted by adipose tissue, either by adipocytes or macrophages/leukocytes. Of note, such obesity-induced inflammation may further contribute to altered insulin and lipid metabolism and atherogenesis in vivo [30].
In addition to its role as central nexus of metabolic communication and control, thermoregulation, and inflammation, this tissue also contributes in the mechanical protection of a series of organs from injury.
In the following sections, anatomical and histological aspects of adipose tissue and relation to physiology, and its relation with dysfunctional adipose during development of obesity will be deeply dissected.
1.2.1 General characteristics of adipose tissue
Adipose tissue can be defined as a type of connective tissue that is mainly composed of adipocytes [5]. Adipocytes are typically found in large aggregates in adipose tissue or “fat” in many organs and different body regions (Figure 2).
Adipose tissue is a dynamic organ which can range from 4% to >40% of total body composition in adult humans [31].
1.2.2 Types of adipose tissue
In mammals, there are two main, visually distinct types of adipose tissue, white adipose tissue (WAT) and brown adipose tissue (BAT) [32].
WAT
In mammals, WAT is usually distributed throughout the body in several distinct discrete anatomical fat depots (Figure 2) [33, 34]. WAT appears generally further sub-classified based on its anatomical location in two major depots, defined as intra-abdominal/visceral (visc)WAT and subcutaneous (sc)WAT. In humans, viscWAT include omental, mesenteric, retroperitoneal (perirenal areas), gonadal, and pericardial WAT. However, it is also found around internal organs and specialized tissues, including vasculature. The other fat depot, the scWAT, is located in different body regions under the skin. In humans, clusters of scWAT can be found in upper (deep and superficial abdomen) and lower (gluteofemoral) body regions [35].
In mice, scWAT is generally located beneath the skin and outside the peritoneal cavity, while viscWAT is located in the abdominal cavity [36]. Although mice and humans share most of fat distribution, they also have distinctive fat depots (Figure 2).
The biology of viscWAT is different from that of scWAT [37]. Different locations suggest that adipose tissue characteristics in each fat depot may differ in its function. Indeed, different WAT depots may be metabolically distinct despite sharing relatively conserved morphology. For instance, viscWAT depots are commonly associated with (cardio)metabolic disorders, including obesity and diabetes [38], whereas scWAT confers beneficial effects on metabolism [39].
Differing metabolic functions of these two major fat depots are extensively reviewed elsewhere [40, 41].
Figure 2 Fat depot distribution in humans and mice.
From [26]
BAT
Histologically, WAT is visually distinct from BAT (Figure 3). In contrast to WAT, BAT is frequently distributed at different locations throughout the body [31]. BAT pads are considerably smaller by volumen and denser than WAT depots, especially in humans, who have lower body surface-to-volume ratio than rodents. Compared with WAT, BAT appears highly vascularized [42].
The largest and most extensively studied BAT depot in rodents, and in most mammals, is located between the shoulder blades (interscapular (i)BAT) and around the kidney (perirenal BAT) as well as in cervical and axillar regions, though it can also be found in other sites such as the para-aortic region, neck and mediastinum [43] (Figure 4). In humans, BAT was thought to be exclusively present in the interscapular region during the perinatal period and adults
chronically exposed to extreme cold; however, recent studies revealed that adult humans have an active BAT [44].
Figure 3 Morphological appearance of BAT and WAT.
Representative mice biopsies of (A) BAT and (B) WAT, fixed with formalin, stained with hematoxylin/eosin and captured at 20X.
The primary main function of BAT is energy dissipation via non-shivering thermoregulation rather than to serve as energy store. Indeed, this tissue uses the triglycerides stored in its numerous lipid droplets as fuel for adaptive, non- shivering thermogenesis [45, 46] and, when fully activated, is able to take up large amounts of fatty acids and glucose from the circulation [46-48].
Figure 4 Distribution of major BAT depots in mouse and human.
From [31]
BAT is highly innervated by the sympathetic nervous system which rapidly triggers brown adipocyte activation upon cold stimulation [46, 49]. In addition to this signal originating from the central nervous system, endocrine and paracrine mediators of brown adipocytes have also been identified, including liver-derived bile acids [50, 51], cardiac natriuretic peptides [52], as well as locally released adenosine [53].
Emerging evidence suggests that mammals possess another type of thermogenic adipocyte (i.e., with characteristics of brown adipocytes) in within WAT depots, defined as ‘beige’ or ‘brite’ adipocytes [54]. However, it is not still clear whether such different designations refer to the same cell type. Indeed, limited genetic tracing experiments suggest that there may be heterogeneity among cell types that are capable of browning. In contrast to brown adipocytes which are located in precisely dedicated BAT depots, beige adipocytes are an inducible form of thermogenic adipocytes that sporadically reside within WAT depots.
1.2.3 Functional units of fat pads
White adipocytes
The accumulation of lipids is characteristic of many cell types; however, the evolution of specialized fat-storing cells, commonly defined as white adipocytes has provided a safe and specific compartment to that effect. White adipocytes are the main cell type present in WAT and are characterized by the presence of a single, large lipid droplet, only a small amount of cytoplasm, and flattened, non-centrally located nuclei, which appear compressed between the lipid droplet and cell membrane; hence they are also defined as ‘unilocular’ adipocytes.
The ‘classical’ function of white adipocytes is to serve as energy store. In conditions of energy excess, free fatty acids released from the hydrolysis of circulating triglycerides, by the action of lipoprotein lipase (LPL), enter adipocytes and are reesterified into triglycerides in a process that involves the sequential action of multiple enzymes, which including glycerol-3-phosphate acyltransferase (G3PAT) and diacylglyceride acyltransferase (DGAT) among others. De novo generated triglycerides accumulate within lipid droplets.
Conversely, in situations of energy deficiency, triglycerides may be mobilized by the action of hormone-sensitive lipase (HSL), adipose tissue triglyceride lipase (ATGL), and monoglyceride lipase (MGL) which releases free fatty acid into circulation.
In addition to the energy-storing properties of white adipocytes, this cell type also has the ability to produce different molecules, including adipokines (adipocyte- derived cytokines), such as leptin, adiponectin and adipsin, and cytokines, such as tumor necrosis factor alpha (Tnfa). The list of adipokines is growing and the understanding of their physiological role on energy balance, cardiovascular function, immune regulation, and nutrient homeostasis exhibit therapeutic potential. However, the specific mechanisms whereby adipokines act on different target tissues still need to be further elucidated.
Brown adipocytes
Brown adipocytes convert chemical energy into heat [55]. Alike white adipocytes, brown adipocytes have multiple lipid droplets of differing size and are rich in mitochondria [7]; hence BAT adopts a brownish color. Nuclei are round and centrally located. Brown adipocytes express a unique thermogenic and mitochondrial genetic program that promotes mitochondrial biogenesis, energy uncoupling and energy dissipation, which provides heat to the organism [55].
Energy dissipation is provided by an abundance of mitochondria as well as by specialized proteins, including a unique protein to thermogenic adipocytes, uncoupling protein-1 (Ucp1). This protein collapses the electron gradient to generate heat rather than ATP [46].
Activation of brown adipocytes with inducers of the brown fat thermogenic program significantly contribute to systemic metabolism by increasing energy expenditure through heat production. The relevance of BAT is exemplified by the fact that in rodents, BAT and liver take up equal amounts of energy from circulation, a process which is able to normalize glucose and lipid values in insulin resistant and hyperlipidemic mice [56].
Beige or ‘brite’ adipocytes
Beige adipocytes are another type of thermogenic fat cell [31]. This cell type may also be present in WAT and exerts a key role in the regulation of systemic energy homeostasis in mammals. In WAT, this class of adipocytes have a remarkably capacity to alter metabolic phenotype from typical WAT to those resembling brown adipocytes [31]. This phenomenon has been defined as ‘browning’ and is readily induced by cold exposure and -adrenergic stimulation. Although browning has been extensively demonstrated only in small mammals, and the extent to which this occurs in humans is still debated. Typically, browning
involves the appearance of multilocular adipocyte clusters that are dispersed among unilocular white adipocytes [31].
Like brown adipocytes, beige adipocytes display thermogenic properties in addition to sharing common morphological and biochemical properties, including multiple lipid droplets and elevated number of mitochondria. However, compelling evidence also suggests distinct characteristics between both types of thermogenic adipocytes. While some brown adipocyte-like express Ucp1 [57, 58], recent evidences may indicate that Ucp1 is not essential for the chronic thermogenic actions of 3-adrenergic agonists, or for the appearance of multilocular fat cells in white fat depots [58, 59]. Supporting to this, their role in the metabolism of glucose may also be accounted for by Ucp1-independent mechanisms [60]. Consistently, Ucp1-negative multilocular adipocytes has been reported to show elevated metabolic rates [61] and PPAR--dependent up- regulation of fatty acid oxidation [62].
Immune cells
Adipocytes are not the only cell type of WAT. This tissue also contains different types of immune cells [5, 63, 64]. Under healthy conditions, the numbers of immune cells are relatively low; however, upon an inflammatory state, as that induced by experimental obesity, immune cell numbers increase rapidly, thereby contributing to metabolic imbalance.
WAT has its own population of resident macrophages, defined as adipose tissue macrophages [65] (Figure 5). This cell type is commonly identified by immunohistochemistry using specific anti-Cd68 antibodies; and hence produce the majority of cytokines from adipose tissue [66, 67]. These cells affect both the metabolic and endocrine functions of adipose tissue [67-69]. Adipose macrophages is actually an heterogeneous population and may be divided into at least two subtypes [70]. Pro-inflammatory, classically-activated macrophages (i.e., M1 macrophages) are associated with immune defense, whereas anti- inflammatory, classically-activated macrophages (i.e., M2 macrophages) are involved in tissue repair and restoration. The ratio between M1/M2 macrophages
is an indicator of the in vivo polarization status of resident macrophages of adipose tissue in response to different stimuli. Pro-inflammatory M1 macrophages accumulate in adipose tissue in obesity [28, 71]. Consistent with this view, macrophages recruited from adipose tissue from diet-induced obesity (DIO) mice have increased inflammatory properties [72]. In contrast, polarization towards anti-inflammatory M2 macrophages preserves insulin sensitivity via PPAR-dependent mechanism [73, 74]. More recently, it has been found that M2 macrophages from adipose tissue also secrete catecholamines, which are involved in increased catabolism and maintaining of thermoregulatory functions in response to cold exposure [75].
Additional immune cell types are present in adipose tissue, though few of them are detected in normal homeostatic conditions. Among them, monocyte, T lymphocyte, leukocyte, neutrophil numbers increase rapidly in WAT in obesity and contribute to the metabolic imbalance of this tissue [5, 63, 64].
Figure 5 Modulation of immune-metabolism during obesity.
Adapted from [76].
1.2.4 Dysfunctional adipose tissue in obesity
When obesity and inflammation are sustained, adaptive homeostatic mechanisms fail, leading to WAT dysfunction [77, 78]. WAT dysfunction generally involves functional changes in adipocytes and macrophages. The term WAT dysfunction is currently used to define abnormal production of pro- atherogenic, pro-inflammatory and pro-diabetic adipokines, which is generally accompanied by a decreased production of adiponectin.
The potential mechanisms involved in adipose tissue dysfunction are heterogeneous and may act directly or in cascade. These include changes in adipose plasticity, ectopic (mainly visceral) fat accumulation, tissue inflammation, and metabolic inflexibility. In addition, tissue cells have the ability to initiate adaptive responses to dysmetabolic stimuli, by plasticity mechanisms, or modification in energy demands, via altering the number, morphology, or remodeling of mitochondria [79]. Indeed, mitochondrial dysfunction may contribute to pathological changes in human tissues in obese.
Adipose plasticity
As a dynamic tissue, WAT may undergo various cellular and structural remodeling processes in response to excess energy and hence the need of energy storage [65]. Tissue remodeling frequently occurs by modifying adipocyte characteristics (i.e., number, size, metabolism). Thus, when tissue expansion is needed, it generally occurs via coordination of [i] enlargement adipocyte size (hypertrophy) and/or number (hyperplasia), [ii] recruitment of inflammatory cells, and [iii] remodeling of the vasculature and the extracellular matrix to allow adequate extracellular expansion, oxygenation and mobilization of nutrients [27].
Similar to WAT, BAT is present at different body sites and can be increased and decreased by environmental signals.
Although adipocyte cellularity is determined early in life, cell turnover remains active throughout life [65]. Interestingly, adipocyte cell turnover is independent of body mass index, which further supports de notion that the adipocyte dotation is early established and is relatively static in adulthood [80, 81]. Apart from homeostatic turnover involved in cell renewal, adipocyte progenitors are activated during in response to signals of hyperplastic expansion. Interestingly, these progenitors can participate in catabolic remodeling, or browning of adipose tissue. Differentiated adipocytes can also contribute to adipose tissue plasticity by undergoing a phenotypic switch between anabolic to catabolic states.
Anatomic location determines whether adipose tissue expansion occurs by hypertrophic or hyperplastic mechanisms in humans and rodents [65].
Remarkably, adipocytes from WAT retain the ability to convert their metabolic phenotype from typical white adipocytes to those resembling brown adipocytes [82-84].
Ectopic fat accumulation
The accumulation of fat deposition in other tissues that regulate metabolic homeostasis, progressive insulin resistance and increases the risk for type 2 diabetes mellitus is referred to as lipotoxicity [85, 86]. Ectopic fat storage during adipose expansion includes extracellular accumulation of fat in intra-abdominal visceral, omental, pericardial, perirenal, and retroperitoneal depots. However, intracellular accumulation of fat in different cell types in organs (i.e., liver, skeletal muscle, heart, pancreas, and kidney) also constitutes a sign of abnormal adipose tissue expansion. Indeed, ectopic accumulation of lipids in the liver, skeletal muscle and pancreas is tightly related to insulin resistance and related diseases, including atherosclerosis [87-91].
Inflammation
Several of the mechanisms involved in adipose dysfunctionality may, either directly or sequentially, induce inflammation. Indeed, obesity is regarded as an inflammatory disease provided by numerous studies showing a moderate increase in circulating pro-inflammatory cytokines in obese patients and the identification of different immune cell types infiltrating the adipose tissue [92].
Rodent and human adipose tissue especially in obese states is the target of macrophage infiltration [28]. Macrophage infiltration proportionally increases with fat mass accumulation and adipocyte hypertrophy [93]. Infiltration of macrophages rises in response to death of hypertrophied adipocytes and aggregate in the form of ‘crown-like structures’, which completely surround adipocytes [94].
In addition to macrophages, the accumulation of T-lymphocytes is also increased in epididymal fat of mice made obese with a high-fat diet and occurs before the accumulation of macrophages [95], thereby suggesting that at least in rodent tissue lymphocyte infiltration might be a primary event in adipose tissue inflammation in obesity.
Neutrophils are the first immune cell type to appear to the inflamed site even before monocyte/macrophage infiltration occurs. Thus, it is tempting to suggest that neutrophil infiltration may precede that of macrophages into ‘inflamed’
adipose tissue. Supporting this view, neutrophils early and transiently infiltrate the parenchyma of intra-abdominal adipose tissue in the course of high-fat feeding in mice [96].
WAT dysfunction is defined by impaired secretion of different adipokines and cytokines [97], thereby contributing to systemic concentrations of pro- and anti- inflammatory cytokines. Indeed, increased numbers of macrophages in human adipose tissue of obese patients are reported to enhance the obesity-related, low-grade chronic inflammation [23]. As secretory cells, resident macrophages may significantly contribute to this phenotype. In WAT dysfunction, the production and secretion of cytokines/adipokines is shifted towards a pro-
inflammatory, atherogenic, and diabetogenic secretion pattern (secreting TNF
and IL-6) in obese (i.e., M1 macrophages). Interestingly, anti-inflammatory mediators, including adiponectin and interleukin (IL)-10, appear reduced in obese states [98], but their systemic levels ameliorate upon excess weight loss interventions (i.e., surgical) [99]. Systemic adiponectin is increased in obese women after body weight reduction in obese women through lifestyle changes is associated with an increase in systemic adiponectin [100]. Conversely, in these conditions systemic IL-10 was not influenced after moderate (diet- induced) weight loss [101].
Metabolic inflexibility
Tissue plasticity was first used as a term to characterize the ability of tissues to respond to a variety of stimuli [102]. Metabolic flexibility is the ability to respond or adapt to changes in metabolic or energy demand. This concept broadly refers to physiological adaptability and it is required to encompass shifts in fuel availability (i.e., glucose and fatty acids) to meet energy demands (i.e., to generate chemical energy and/or key metabolites). Insulin release is a major driver of this shift as it governs metabolic machinery to move from catabolic to anabolic processes to store energy.
Metabolic flexibility appears altered in obesity and type 2 diabetes, when fuel selection fails [102]. In the context of obesity, insulin resistance would be an example of metabolic inflexibility, i.e., dysfunctional response to insulin- mediated stimulation, and can develop in many tissues and organs, including WAT.
Despite the analysis of metabolic flexibility in WAT is poorly explored; several reports suggest that this tissue also plays an active role in the metabolism of glucose and lipids, as well as having the potential to increase thermogenesis (i.e., browning) [103-105]. As previously indicated, WAT buffers circulating free fatty acids for peripheral tissues such as the liver via a fine-tuned system that
allows their uptake, esterification, and release [106]. Importantly, this process also occurs in BAT [107], though metabolic flexibility of BAT involves intracellular triglyceride combustion to obtain energy rather than storage and supply for peripheral tissues, as is the case of WAT.
Excess of WAT (i.e., obesity) is related to metabolic complications. However, the mass of WAT is not itself the only cause to explain obesity-related complications, which highlights the importance of healthy, metabolically adaptable WAT to explain them. In this regard, free fatty acid levels (coming from an inappropriate lipolysis) represent an etiological factor for insulin resistance and type 2 diabetes mellitus [108, 109]. In humans, the only significant site of free fatty acid release is abdominal scWAT [110]. Insulin-induced suppression of triglyceride lipolysis in WAT is disturbed in the type 2 diabetes mellitus [111], which put emphasis in the sensitivity (flexibility) of WAT to insulin in a healthy state and reveals the potential of this tissue, which is prone to blunted insulin responsiveness, as an early aberration in the etiology of whole-body insulin resistance and type 2 diabetes mellitus.
The metabolic adaptability of WAT in obesity to expand and continuously store free fatty acids apparently inertly as its ‘classical’ function, which is down- regulated in conditions of obesity-driven insulin resistance of basal (fasting) [112, 113] and dietary fat storage [114]. Expandibility of WAT is limiting.
Thiazolidinediones (TZDs) markedly improve insulin sensitivity and glucose metabolism by expanding WAT via PPAR activation and improving triglyceride metabolism [115].
The potential usefulness of metabolic flexibility as viable targets for preventing or treating obesity and type 2 diabetes mellitus is controversial. The effectiveness of these therapies flaw in conditions whereby fuel selections appears altered, but in the absence of concomitant increases in energy demand, as it occurs in nutrient overload or obesity [116]. Indeed, increasing mitochondrial fatty acid flux and oxidation may [117] or may not [118] improve insulin resistance. Taken together, strategies against metabolic diseases such as obesity will be effective only whether they exert a concomitant increase in energy expenditure or demand (like exercise).
Mitochondrial dysfunction
Mitochondria play a central role in metabolism in adipose tissue [119]. They deeply contribute to lipolysis and lipogenesis pathways in white adipocytes, and weight control is now widely accepted [120].
Mitochondrial dysfunction may result from a decreased mitochondrial biogenesis, reduced mitochondrial content, and/or a decrease in the protein content and activity of oxidative proteins ‘per unit of mitochondria’ [121].
Although the main tissues affected by mitochondrial dysfunction are those with high energy demand, including skeletal muscle, heart, brain; accumulating evidence targeting mitochondria in adipocytes or adipose tissues strongly suggests that mitochondrial impairment in adipose tissue from obese also contribute to whole-body pathological consequences [120, 122]. However, this is not still clear whether mitochondrial dysfunction plays a causative or adaptive role in various metabolic conditions related to obesity or type 2 diabetes mellitus.
Compelling lines of evidence indicate that major factors contributing to mitochondrial defects in adipose tissues are oxidative stress, insulin resistance, genetic factors and sedentary lifestyle without physical activity [123].
- Oxidative stress
Oxidative stress has been implicated in the pathogenesis of obesity [124].
Oxidative stress is defined by a disequilibrium between the production of ROS and antioxidant defence [125]. Mitochondria represent the major source of intracellular free radicals. Excess ROS species may damage proteins, lipids, and DNA in cells. Defects in the transfer of electrons across the mitochondrial membrane as results of their accumulation in the respiratory chain complexes, enhances their binding with free oxygen, and hence ROS production [126].
Increases in ROS may be also caused by elevated levels of fatty acids via NADPH oxidase activation in adipocytes [127]. In this scenario, ROS may contribute to abnormal production rates of different adipokynes, including
adiponectin. In obese mice, fat accumulation correlates with systemic oxidative stress and treatment with inhibitors of NADPH oxidase reduces ROS production in adipose tissue, attenuated adipokine dysregulation, and improved diabetes, hyerlipidemia, and hepatic steatosis in humans and mice [127]. Moreover, increases in intracellular ROS levels elicited by mitochondrial dysfunction promotes adipocyte dysfunction in the maintenance of glucose homeostasis through attenuation of insulin signalling, down-regulation of glucose transporter- (Glut)4 expression, and decrease in adiponectin secretion [128].
- Insulin resistance
Insulin resistance is a metabolic defect associated with obesity [129], which consists of an dysfunctional action of insulin to produce its effects on glucose, protein and lipid metabolism in target tissues. Decreased insulin response to glucose, dislipidemia, and obesity frequently progress into Type 2 diabetes mellitus with a decline in the -cell function, sustained hyperglycemia and increased advanced glycation end products (AGE) formation. AGE may in turn contribute to insulin resistance in adipose tissue of obese [130]. The contribution of mitochondria in adipose tissues in the onset and progression of insulin resistance is controversial. Recent data suggest a role for an altered flux of mitochondrial calcium in impairing insulin sensitivity [131] that is associated with impaired mitochondrial biogenesis and decreased expression of mitochondrial proteins in adipose tissue [132, 133]. However, ROS-induced mitochondrial dysfunction may be a valid mechanism in skeletal muscle, but not in adipocytes [134], suggesting that it may be tissue-specific.
- Genetic factors
There is a spectrum of genes and/or gene variants linked to mitochondrial function and mass in adipose tissue positively related to obesity or obesity- related phenotypes [135]. Moreover, altered gene expression of molecular
targets controlling mitochondrial activity and biogenesis in adipose tissue may be caused by mutations of both nuclear and mitochondrial genomes [124, 136]
as well as defects in the gene expression mitochondria-related genes in adipocytes [132, 137, 138]. In this context, transcriptional activators such as Pgc1a and Pgc1b play a crucial role in coordinating the gene expression of mitochondrial and nuclear genes related to mitochondrial metabolism in both WAT and BAT [139, 140]. Pgc1 enhances PPAR in human WAT toward a transcriptional program linked to energy dissipation through an increased expression of Ucp1 [141]. Consistent with this view, gene expression of Pgc1a is reduced in scWAT of morbidly obese patients [142]. Although it is still pending to elucidate whether decreases in the expression of Pgc1a can lead to development of obesity or just a consequence of it, the up-regulation of thermogenic genes in WAT might offer new therapeutic tools for obesity.
- Changes in human behavior and lifestyle
Sedentary lifestyle have a dramatic impact on human health [143, 144]. Exercise is a major modulator of mitochondrial function [145, 146]. Indeed, the ‘beiging’
of WAT concomitant to induced gene expression of Ucp1 (specific of brown adipocytes) has been shown in WAT in response to exercise training [147].
Supporting to this, elevations in the mRNA levels of Pgc1a has been found in WAT of mice subjected an acute exercise training [148].
▶ 1.3 Obesity and cardiovascular disease
Compelling clinical studies suggest that obesity is an independent risk factor for cardiovascular disease [149]. This co-morbidity is frequently associated with an altered metabolic profile, defined as metabolic syndrome [108, 109]. Besides metabolic syndrome, excess adipose tissue accumulation elicits a spectrum of cardiovascular adaptations in obese patients [150, 151].
Coronary artery disease (CAD) represents one of the main outcomes of cardiovascular disease [152]. Atherosclerosis development is the pathogenic mechanism underlying CAD. Atherosclerosis progression typically begins as results of accumulating cholesterol deposits in monocyte-derived macrophage foam cells in the intima of large arteries (fatty streaks) at early stages of human life [153-155]. Endothelial dysfunction in epicardial vessels and inflammation in the vessel wall are two of the main early events involved in its progression.
Obese patients are at high risk for CAD [152]. Atherosclerosis can be assessed through determining the carotid intimal-medial thickness (IMT), a marker of generalized atherosclerosis. Despite its limitations [156, 157], this carotid IMT has been linked to obesity [158-161].
Adipose tissue synthesizes and releases to circulation a variety of molecules with a role in cardiovascular homeostasis. Indeed, this tissue is a significant source of tumor necrosis factor-alpha (Tnf-) and interleukin-(IL)6 as well as other adiponectins [162-165]. Particularly, adipose tissue is a main contributor of total circulating IL-6 levels, with around 30% of this cytokine coming from this tissue [166, 167]. Of clinical consideration, this finding is very important since IL- 6 modulates hepatic synthesis and release of CRP, a marker of chronic inflammation, which can trigger acute coronary syndrome [168].
On a clinical context, circulating concentrations of PAI-1, C-reactive protein (CRP), fibrinogen, and Tnf- are all related to body mass index (BMI) [166, 169].
However, other surrogates of body weight, such as waist-to-hip ratio and waist circumference are also good indicators of abdominal obesity, and found to be more closely related to atherosclerosis than BMI [170].
